The central nervous system (CNS) is particularly vulnerable to insults that result in cell death or damage in part because cells of the CNS have a limited capacity for repair. As a result, disorders of the CNS often result in debilitating and largely irreversible degradation of a patient""s cognitive and sensorimotor functions. Conditions that result in nerve cell death and damage range from degenerative disorders, such as Alzheimer""s disease, to ischemic episodes, such as stroke, to trauma.
Injury to the central nervous system (CNS) is an important cause of death and disability worldwide. For example, stroke is the third leading cause of death and disability in the U.S., with an estimated incidence of 700,000 cases annually (Furie et al. (1998) xe2x80x9cCerebrovascular Diseasexe2x80x9d in The Atlas of Clinical Neurology, R. N. Rosenberg, Ed. Current Medicine: Philadelphia). Two-thirds of stroke patients survive the first year following stroke, for an average of seven years, leading to more than 4.8 million stroke survivors currently in the U.S. Stroke costs the U.S. economy in excess of $30 billion per year in terms of medical costs and lost wages.
After several hours, little can be done to prevent the direct damage to the CNS caused by CNS disorders. For example, stroke treatments must typically be administered within six hours of onset. Depending on where the injury occurs in the brain, patients may be paralyzed on one side, may lose the ability to speak or see, and may have difficulty walking, among other symptoms. Gradual recovery of these functions is common, although recovery may be incomplete, and depends on the size and location of injury, among other factors.
Since damaged brain tissue does not regenerate, recovery must come from the remaining intact brain, which reorganizes itself, or rewires, in order to compensate for some of the function lost by the damage. Indeed, studies in animals and humans provide ample evidence of such reorganization of brain function following stroke. In particular, remaining neurons in both the damaged hemisphere and in the opposite intact hemisphere grow new processes (both axons and dendrites) and form new connections (synapses), which most likely contribute to recovery (Kawamata et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 8179-8184; Jones et al. (1994) J. Neurosci., 14: 2140-2152; Stroemer et al. (1998) Stroke, 29: 2381-2395; Cramer et al. (1997) Stroke, 28: 2518-2527).
As an example, stroke treatment has focused on limiting the extent of damage within the first few hours. Stroke is generally due to a blockage of an artery leading to the brain, resulting in the death of brain cells supplied by that artery. Current treatments for stroke have centered on treatments to prevent arterial blockages (control of blood pressure, lipids, heart disease, etc.), and treatments to prevent brain damage once the blockage has occurred. These latter treatments include xe2x80x9cthrombolytic agentsxe2x80x9d (xe2x80x9cclot bustersxe2x80x9d such as tPA) to break up arterial clots, and xe2x80x9cneuroprotective agents,xe2x80x9d designed to protect brain tissue at risk for stroke. Such thrombolytic and neuroprotective agents must be administered within hours after the onset of stroke in order to be effective.
Currently there are only a few available methods of promoting recovery in patients after cell death and injury has already occurred. Methods of treating stroke after the initial phase of damage are mechanistically different from methods used in the first few hours. Treatments to promote recovery typically focus on encouraging neuronal growth and rewiring.
Direct application of neurotrophic growth factors to the brain can enhance spontaneous functional recovery occurring in animal models of stroke (Kawamata et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 8179-8184; Kawamata et al. (1996) J. Cereb. Blood Flow Metab., 16: 542-547; Kawamata et al. (1999) Exp. Neurol. 158: 89-96; Alps et al., U.S. Pat. No. 5,733,871, Fisher et al. (1995) J. Cereb. Blood Flow Metab., 15: 953-959; Jiang et al. (1996) J. Neurol. Sci., 139: 173-179). For example, basic fibroblast growth factor (bFGF) is a protein that supports survival and axonal outgrowth from neurons. When bFGF is administered starting a day or more after stroke, animals recover more quickly and to a greater extent on tests of sensorimotor function of the impaired limbs (opposite to the side of the stroke). This recovery is not due to a decrease in magnitude of the original brain damage. Instead, data suggests that this enhancement of recovery may be due to enhancement of new neuronal sprouting and synapse formation in the intact remaining brain tissue. Such remodeling appears to occur in both the damaged and undamaged hemispheres. Other mechanisms of recovery may include stimulation of endogenous neural stem cells within the brain that then differentiate into neurons, replacing to some extent neurons lost by stroke.
Another potential approach to a treatment for stroke recovery includes the use of neural stem cells. These are pluripotential cells already present in the developing and mature mammalian brain that, given the appropriate stimulation, can differentiate into brain neurons and/or glial cells. Several investigators have been successful in separating and cloning out such neural stem cell lines from both the murine and human brain (Snyder et al. (1997) Proc. Natl. Acad. Sci. USA, 94: 11663-11668; Gage et al. (1995) Proc. Natl. Acad. Sci. USA, 92: 11879-11883; Kuhn et al. (1997) J. Neurosci., 17: 5820-5829; McKay et al., U.S. Pat. No. 5,270,191; Johe, K., U.S. Pat. No. 5,753,506; Carpenter, M., U.S. Pat. No. 5,968,829; Weiss et al., U.S. Pat. No. 5,750,376). When such stem cells are reintroduced into the developing or mature brain, they can divide, migrate, grow processes, and assume neural phenotypes, including the expression of neurotrarsmitters and growth factors normally elaborated by neurons. Thus, use of neural stem cells may be advantageous for stroke recovery in at least two ways: (1) by the stem cells partially repopulating dead areas and re- establishing neural connections lost by stroke, and (2) by secretion of important neurotrarsmitters and growth factors required by the brain to rewire after stroke. Efforts to promote recovery from brain injury in animals using neural stem cells have been described (Park et al. (1999) J. Neurotrauma 16: 675-687; Park et al. (1995) Soc. Neurosci. Abs. 21: 2027; Stroemer et al. (1999) Soc. Neuroscience Abs. 25:1310). Efforts using a line of teratocarcinoma-derived cells have also been described in animals (Borlongan et al. (1993) Int. J. Devl. Neuroscience 11: 555-568) and humans (Kokaia et al. (1998) Eur. J. Neurosci., 10: 2026-36).
Methods currently available for promoting recovery from CNS damage allow only partial recovery of neurological functions. In patients suffering from debilitating neurological deficits, incremental improvements in function may have a significant effect on quality of life. Given the large number of affected patients and the limitations of current methods, there is an urgent need for additional and improved methods to promote recovery from damage to the nervous system. The modes of treatment presented herein promote a greater degree of recovery from CNS damage than is currently available with other known treatment methods.
One aspect of the present application relates to methods for improving a subject""s recovery from CNS injury or damage. In one aspect, the invention comprises administering to a subject cells, preferably stem cells, and a neural stimulant in sufficient amounts to improve the subject""s sensorimotor or cognitive abilities, e.g., improved limb movement and control or improved speech capability.
In another aspect, the invention provides kits for the treatment of CNS damage. In certain embodiments, kits of the invention comprise stem cells and a neural stimulant. In other embodiments, the kits of the invention comprise a neural stimulant and a device for obtaining a stem cell-containing sample from a subject. In preferred embodiments, the kits comprise a polypeptide growth factor, and more preferably a polypeptide at least 30% identical but most preferably at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% identica of the polypeptides of SEQ. ID. Nos. 1-3.
In a further aspect, the invention provides pharmaceutical preparations comprising stem cells, a neural stimulant and one or more pharmaceutically acceptable reagents.
In preferred embodiments, stem cells for use in the invention are cells capable of giving rise to brain cells, eg. neurons, oligodendroglia or astroglia. In particularly preferred embodiments, stem cells are neural stem cells, hematopoietic stem cells, teratocarcinoma-derived cells or embryonic stem cells. In other preferred embodiments, stem cells are obtained from the subject, and optionally cultured or enriched in vitro prior to administration.
In other embodiments, stem cells of the invention may be induced to proliferate in vitro by transfection with a gene encoding one or more proliferation promoting factors, such as vmyc, SV40 T antigen, polyoma virus large T antigen, the neu oncogene or the ras oncogene. In preferred embodiments, the gene is strongly expressed in vitro, promoting proliferation, and poorly expressed after the cell has entered the central nervous system, such that the cell does not proliferate rapidly in vivo.
In a further embodiment, the neural stimulant is a polypeptide growth factor. Preferred polypeptide growth factors comprise a polypeptide that is chosen from among the following polypeptide families: fibroblast growth factor family members, neurotrophin family members, insulin-like growth factor family, ciliary neurotrophic growth factor family members; EGF family members, TGFxcex2 family members, leukemia inhibitory factor (LIF); oncostatin M, interleukin-11; interleukin-6; members of the platelet-derived growth factor family, and VEGF family members. It is contemplated that, in certain embodiments, combinations of factors may be used. Preferred polypeptides comprise a polypeptide with a sequence that is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% percent identical to an amino a sequence shown in any of SEQ ID Nos. 1-3.
In still other embodiments, the neural stimulant is a modulator of neurotrarsmitter activity (eg. an agonist or antagonist). In preferred embodiments, the neural stimulant is an antidepressant, such as Prozac, an amphetamine, Ritalin, a tricyclic antidepressant such as Elavil, or combinations thereof. In another embodiment, the neural stimulant is a promoter of neuronal differentiation such as retinoic acid. In yet another embodiment, the neural stimulant is a so-called guidance molecule such as a netrin, a semaphorin, a neuropilin or an ephrin. In yet an additional embodiment, the neural stimulant may be transcranial magnetic stimulation.
In another aspect, the invention comprises conjoint administration of cells with a bioactive compound that is not a neural stimulant. Preferred bioactive compounds include immunosuppressants such as immunophilins (eg. cyclosporin, FK506, and thalidomide) and antibiotics, such as tetracycline.
A range of techniques for administering the cells and neural stimulants of the invention are contemplated. Cells and neural stimulants do not need to be administered in the same way or at the same time, but they are preferably administered such that their effects overlap. In preferred embodiments, administration is carried out at least 6, 10, 12 or 24 hours after the injury has occurred.